Polypeptide-calcitonin 11 and the polynucleotide encoding it

ABSTRACT

The present invention discloses a new polypeptide-calcitonin 11, the polynucleotide encoding it and a method producing the polypeptide by recombinant DNA technology. The present invention further discloses a method using the polypeptide to treat various disorders, e.g. malignant neoplasm, hematopathy, HIV infection and immunological disease and various inflammation etc. The present invention also discloses agonists of the polypeptide and their therapeutic uses. The present invention further discloses the use of the polynucleotide encoding the new calcitonin 11.

FIELD OF INVENTION

[0001] The invention relates to the field of biotechnology. Inparticular, the invention relates to a novel polypeptide, calcitonin 11,and a polynucleotide sequence encoding the polypeptide. The inventionalso relates to the method for the preparation and use of thepolynucleotide and polypeptide.

TECHNICAL BACKGROUND

[0002] Calcitonin, calcitonin Gene-related peptide (CGRP) and isletamyloid polypeptide (IAPP) have similarities in their structure.Calcitonin is a polypeptide hormone with 32 amino acid residues. Bybinding to the calcium ion, it can modulate the concentration of calciumion in blood (Breimer, et al., 1988, Biochem. J. 255:377-390). CGRP is apolypeptide hormone with 39 amino acid residues. It can causevasodilatation in different vessels (including coronary artery, bloodvessel of brain, and the entire vascular system). IAPP is a polypeptidehormone with 37 amino acid residues. It can selectively inhibit glucosemetabolism and glycogen synthesis which are induced by insulin (Nishi etal., 1990, J. Biol. Chem. 265: 4173-4176).

[0003] In almost all organisms, the three proteins share a conservedstructural motif: C-[SAGDN]-[STN]-x (0,1)-[SA]-T-C-[VMA]-x (3)-[LYF]-x(3)-[LYF], and the two cysteines was bonded by a disulfide bond.

[0004] The conserved structural motif found in those three proteins isshown below:

[0005] where * denotes conserved amino acid residues, C denotes theconserved cysteine. There are two conserved cysteines in the N-terminal, and the C-terminal of the protein was amidated. This conservedmotif apparently plays a critical role to the physiological functions ofcalcitonin, CGRP and IAPP.

[0006] It is known that calcitonin is essential in the modulation ofcalcium concentration in the blood. It is used to treat infanthyperglycemia of unknown causes, and adult hypercalcimia caused byhyperparathyroidism. Calcitonin is also important in preventing loss ofbone mass and decrease in bone density. Calcitonin deficiency will causeosteoporosis (BMJ, 1989; 298: 1215-6; Biochem J Oct. 15, 1988; 255:377-90).

[0007] Research on the effect of CGRP on the brain and other tissuesshowed that CGRP can cause vasodilaiton, and it is an importantregulator of blood flow and blood vessel tension. Furthermore, CGRP isimportant in the control of certain nervous system functions, actions ofhuman cell and regulation of the endocrine system.

[0008] As discussed above, calcitonin plays an important role theregulation of many biological processes, which regulation is believed torequire numerous proteins. It is, therefore, very important to identifyadditional proteins involved in the regulatory process. The isolation ofthe calcitonin 11 gene of the present invention enables theidentification of its function under healthy and diseased conditions.

Disclosure of Invention

[0009] One objective of the invention is to provide an isolated novelpolypeptide, i.e., a calcitonin 11, and fragments, analogues andderivatives thereof.

[0010] Another objective of the invention is to provide a polynucleotideencoding the polypeptide.

[0011] Another objective of the invention is to provide a recombinantvector containing a polynucleotide encoding a calcitonin 11.

[0012] Another objective of the invention is to provide a geneticallyengineered host cell containing a polynucleotide encoding a calcitonin11.

[0013] Another objective of the invention is to provide a method forproducing a calcitonin 11.

[0014] Another objective of the invention is to provide an antibodyagainst a calcitonin 11 of the invention.

[0015] Another objective of the invention is to provide mimetics,antagonists, agonists, and inhibitors for the polypeptide of thecalcitonin 11.

[0016] Another objective of the invention is to provide a method for thediagnosis and treatment of diseases associated with an abnormality ofcalcitonin 11.

[0017] The present invention relates to an isolated polypeptide, whichis originated from human, and comprises a polypeptide having the aminoacid sequence of SEQ ID NO: 2, or its conservative variants, or itsactive fragments, or its active derivatives and its analogues.Preferably, the polypeptide has the amino acid sequence of SEQ ID NO: 2.

[0018] The present invention also relates to an isolated polynucleotide,comprising a nucleotide sequence or its variant selected from the groupconsisting of (a) the polynucleotide encodeing a polypeptide comprisingthe amino acid sequence of SEQ ID NO: 2; and (b) a polynucleotidecomplementary to the polynucleotide (a); (c) a polynucleotide thatshares at least 70% homology to the polynucleotide (a) or (b).Preferably, the nucleotide sequence is selected from the groupconsisting of (a) the sequence of position 211-501 in SEQ ID NO: 1; and(b) the sequence of position 1-1614 in SEQ ID NO: 1.

[0019] The invention also includes: a vector comprising a polynucleotideof the invention, especially an expression vector; a host cellgenetically engineered with the vector via transformation, transductionor transfection; a method for the production of the inventivepolypeptide through the process of host cell cultivation and expessionproduct harvest.

[0020] The invention also relates to an antibody which specificallybinds to the inventive polypeptide.

[0021] The invention also relates to a method for selecting compoundswhich could simulate, activate, antagonize, or inhibit the activity ofthe inventive polypeptide and the compounds obtained by the method.

[0022] The invention also relates to a method for in vitro diagnosis ofdiseases or disease susceptibility related with the abnormal expressionof the inventive polypeptide. The method involves the detection ofmutation in the polypeptide or its encoding polynucleotide sequence, orthe determination of its quantity and/or biological activity inbiological samples.

[0023] The invention also relates to pharmaceutical compositions whichcomprises the inventive polypeptide, its analogues, mimetics, agonists,antagonists, inhibitors, and a pharmaceutically acceptable carrier.

[0024] The invention also relates to applications of the inventivepolypeptide and/or its polynucleotide for drug development to treatcancers, developmental diseases, immune diseases, or other diseasescaused by abnormal expression of the inventive polypeptide.

[0025] Other aspects of the invention are apparent to the skilled in theart in view of the disclosure set forth hereinbelow.

[0026] The terms used in this specification and claims have thefollowing meanings, unless otherwise noted.

[0027] “Nucleotide sequence” refers to oligonucleotide, nucleotide, orpolynucleotide and parts of polynucleotide. It also refers to genomic orsynthetic DNA or RNA, which could be single stranded or double stranded,and could represent the sense strand or the antisense strand. Similarly,the term “amino acid sequence” refers to oligopeptide, peptide,polypeptide, or protein sequence and parts of proteins. When the “aminoacid sequence” in the invention is related to the sequence of a naturalprotein, the amino acid sequence of the “peptide” or “protein” will notbe limited to be identical to the sequence of that natural protein.

[0028] “Variant” of a protein or polynucleotide refers to the amino acidsequence or nucleotide sequence, respectively with one or more aminoacids or one or more nucleotides changed. Such changes include deletion,insertion, and/or substitution of amino acids in the animo acidsequence, or of nucleotides in the polynucleotide sequence. In the caseof polypeptides, these changes could be conservative and the substitutedamino acid has similar structural or chemical characteristics as theoriginal one, such as the substitution of Ile with Leu. Changes alsocould be not conservative, such as the substitution of Ala with Trp.

[0029] “Deletion” refers to the deletion of one or several amino acidsin the amino acid sequence, or of one or several nucleotides in thenucleotide sequence.

[0030] “Insertion” or “addition” refers to the addition of one orseveral amino acids in the amino acid sequence, or of one or severalnucleotides in the nucleotide sequence, comparing to the naturalmolecule. “Substitution” refers to the change of one or several aminoacids, or of one or several nucleotides, into different ones withoutchanging number of the residues.

[0031] “Biological activity” refers to structural, regulatory orbiochemical characteristics of a molecule. Similarly, the term“immunogenecity” refers to the ability of natural, recombinant, orsynthetic proteins or other molecules to induce a specific immunereaction in an appropriate animal or cell, or to bind to a specificantibody.

[0032] “Agonist” refers to molecules which regulate, but generallyenhance the activity of the inventive polypeptide by binding to and/orchanging it. Agonists include proteins, nucleotides, carbohydrates orany other molecules which could bind to the inventive polypeptide.

[0033] “Antagonist” or “inhibitor” refers to molecules which inhibit ordownregulate a biological activity or immunogenecity of the inventivepolypeptide via binding to it. Antagonists or inhibitors includeproteins, nucleotides, carbohydrates or any other molecules which bindto the inventive polypeptide.

[0034] “Regulation” refers to changes in the function of the inventivepolypeptide, including up-regulation or down-regulation of the proteinactivity, changes in binding specifity, changes of any other biologicalcharacteristics, functional or immune characteristics.

[0035] “Substantially pure” refers to the condition of substantiallyfree of other naturally related or associated proteins, lipids,saccharides, or other substances. One of ordinary skill in the art canpurify the inventive polypeptide by standard protein purificationtechniques. Substantially pure polypeptide of the invention produces asingle main band in a denaturing polyacrylamide gel. The purity of apolypeptide may also be analyzed by amino acid sequence analysis.

[0036] “Complementary” or “complementation” refers to the binding ofpolynucleotides by base pairing under approximate ion and temperatureconditions. For instance, the sequence “C-T-G-A” could bind to itscomplementary sequence “G-A-C-T.” The complementation between two singlestrand molecules could be partial or complete. Homology or sequencesimilarity between two single strands obviously influences theefficiency and strength of the formed hybrid.

[0037] “Homology” refers to the complementary degree, which may bepartial or complete. “Partial homology” refers to a sequence beingpartially complementary to a target nucleotide. The sequence could atleast partially inhibit the hybridization between a completelycomplementary sequence and the target nucleotide. Inhibition ofhybridization could be assayed by hybridization (Southern blot orNorthern blot) under less stringent conditions. Substantiallycomplementary sequence or hybrid probe could compete with the completelycomplementary sequence and inhibit its hybridization with the targetsequence under less stringent conditions. This does not mean thatnonspecific binding is allowed under a less stringent condition, becausespecific or selective reaction is still required.

[0038] “Sequence Identity” refers to the percentage of sequence identityor similarity when two or several amino acid or nucleotide sequences arecompared. Sequence identity may be determined by computer programs suchas MEGALIGN (Lasergene Software Package, DNASTAR, Inc., Madison Wis.).MEGALIGN can compare two or several sequences using differentmethodologies such as the Cluster method (Higgins, D. G. and P. M.Sharp, 1988, Gene 73:237-244). Cluster method examines the distancebetween all pairs and arrange the sequences into clusters. Then theclusters are partitioned by pair or group. The sequence identity betweentwo amino acid sequences such as sequence A and B can be calculated bythe following equation:$\frac{{Number}\quad {of}\quad {paired}\quad {identical}\quad {residues}\quad {between}\quad {sequences}\quad A\quad {and}\quad B}{{{Residue}\quad {number}\quad {of}\quad {sequence}\quad A} - {{number}\quad {of}\quad {gap}\quad {residues}\quad {in}\quad {sequence}\quad A} - {{number}\quad {of}\quad {gap}\quad {residue}{\quad \quad}{in}\quad {sequence}\quad B}} \times 100$

[0039] Sequence identity between nucleotide sequences can also bedetermined by Cluster method or other well-known methods in the art suchas the Jotun Hein method (Hein J., 1990, Methods in Emzymology183:625-645)

[0040] “Similarity” refers to the degree of identity or conservativesubstitution degree of amino acid residues in corresponding sites of theamino acid sequences when compared to each other. Amino acids forconservative substitution are: negative charged amino acids includingAsp and Glu; positively charged amino acids including Leu, Ile and Val;Gly and Ala; Asn and Gln; Ser and Thr; Phe and Tyr.

[0041] “Antisense” refers to the nucleotide sequences complementary to aspecific DNA or RNA sequence. “Antisese strand” refers to the nucleotidestrand complementary to the “sense strand.”

[0042] “Derivative” refers to the inventive polypeptide orpolynucleotide chemically or otherwise modified. This kind of modifiedchemical may be derived from replacement of the hydrogen atom with analkyl, acyl, or amino group. The nucleotide derivative can encodepeptide retaining the major biological characteristics of the naturalmolecule.

[0043] “Antibody” refers to the intact antibody or its fragments such asFa, F (ab′)2 and Fv, and it can specifically bind to antigenic epitopesof the inventive polypeptide.

[0044] “Humanized antibody” refers to an antibody which has its aminoacid sequence in non-antigen binding region replaced to mimic humanantibody and still retain the original binding activity.

[0045] The term “isolated” refers to the removal of a material out ofits original environment (for instance, if it's naturally produced,original environment refers to its natural environment). For example, anaturally produced polynucleotide or a polypeptide in its original hostorganism means it has not been “isolated,” while the separation of thepolynucleotide or a polypeptide from its coexisting materials in naturalsystem means it was “isolated.” This polynucleotide may be a part of avector, or a part of a compound. Since the vector or compound is notpart of its natural environment, the polynucleotide or peptide is still“isolated.”

[0046] As used herein, the term “isolated” refers to a substance whichhas been isolated from the original environment. For naturally occurringsubstance, the original environment is the natural environment. Forexample, the polynucleotide and polypeptide in a naturally occurringstate in the viable cells are not isolated or purified. However, if thesame polynucleotide and polypeptide have been isolated from othercomponents naturally accompanying them, they are isolated or purified.

[0047] As used herein, “isolated calcitonin 11,” means that calcitonin11 does not essentially contain other proteins, lipids, carbohydrate orany other substances associated therewith in nature. The skilled in theart can purify calcitonin 11, by standard protein purificationtechniques. The purified polypeptide forms a single main band on anon-reducing PAGE gel. The purity of calcitonin 11 can also be analyzedby amino acid sequence analysis.

[0048] The invention provides a novel polypeptide—calcitonin 11, whichcomprises the amino acid sequence shown in SEQ ID NO: 2. The polypeptideof the invention may be a recombinant polypeptide, natural polypeptide,or synthetic polypeptide, preferably a recombinant polypeptide. Thepolypeptide of the invention may be a purified natural product or achemically synthetic product. Alternatively, it may be produced fromprokaryotic or eukaryotic hosts, such as bacterial, yeast, higher plant,insect, and mammal cells, using recombinant techniques. Depending on thehost used in the protocol of recombinant production, the polypeptide ofthe invention may be glycosylated or non-glycosylated. The polypeptideof the invention may or may not comprise the starting Met residue.

[0049] The invention further comprises fragments, derivatives andanalogues of calcitonin 11. As used in the invention, the terms“fragment,” “derivative” and “analogue” mean the polypeptide thatessentially retains the same biological functions or activity ofcalcitonin 11 of the invention. The fragment, derivative or analogue ofthe polypeptide of the invention may be (i) one in which one or more ofthe amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code; or (ii) one in which one or more of theamino acid residues are substituted with other residues, including asubstituent group; or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol); or (iv) one in whichadditional amino acids are fused to the mature polypeptide, such as aleader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthe skilled in the art from the teachings herein.

[0050] The invention provides an isolated nucleic acid or polynucleotidewhich comprises the polynucleotide encoding an amino acid sequence ofSEQ ID NO: 2. The polynucleotide sequence of the invention includes thenucleotide sequence of SEQ ID NO: 1. The polynucleotide of the inventionwas identified in a human embryonic brain cDNA library. Preferably, itcomprises a full-length polynucleotide sequence of 1614 bp, whose ORF(211-501) encodes a polypeptide of 96 amino acids, which has thecharacteristic sequence of the calcitonin family of proteins, andsimilar structures and functions as well.

[0051] The polynucleotide according to the invention may be in the formsof DNA or RNA. The forms of DNA include cDNA, genomic DNA, and syntheticDNA, etc., in single stranded or double stranded form. DNA may be acoding strand or a non-encoding strand. The coding sequence for maturepolypeptide may be identical to the coding sequence shown in SEQ ID NO:1, or is a degenerate sequence. As used herein, the term “degeneratesequence” means a sequence which encodes a protein or peptide comprisinga sequence of SEQ ID NO: 2 and which has a nucleotide sequence differentfrom the sequence of coding region in SEQ ID NO: 1.

[0052] The polynucleotide encoding the mature polypeptide of SEQ ID NO:2 includes those encoding only the mature polypeptide, those encodingmature polypeptide plus various additional coding sequence(s), thecoding sequence for mature polypeptide (and optional additional encodingsequence) plus the non-coding sequence(s).

[0053] The term “polynucleotide encoding the polypeptide” includespolynucleotides encoding the polypeptide and polynucleotides comprisingadditional coding and/or non-coding sequences.

[0054] The invention further relates to variants of the abovepolynucleotides which encode a polypeptide having the same amino acidsequence of the invention, or a fragment, analogue and derivative of thepolypeptide. The variant of the polynucleotide may be a naturallyoccurring allelic variant or a non-naturally occurring variant. Suchnucleotide variants include substitution, deletion, and insertionvariants. As known in the art, an allelic variant may have asubstitution, deletion, and insertion of one or more nucleotides withoutsubstantially changing the functions of the encoded polypeptide.

[0055] The present invention further relates to polynucleotides, whichhybridize to the hereinabove-described sequences, that is, there is atleast 50% and preferably at least 70% identity between the sequences.The present invention particularly relates to polynucleotides, whichhybridize to the polynucleotides of the invention under stringentconditions. As herein used, the term “stringent conditions” means thefollowing conditions: (1) hybridization and washing under low ionicstrength and at a high temperature, such as 0.2×SSC, 0.1% SDS, 60° C.;or (2) hybridization after adding denaturants, such as 50% (v/v)formamide, 0.1% bovine serum/0.1% Ficoll, 42° C.; or (3) hybridizationonly when the homology of two sequences at least 95%, preferably 97%.Further, the polynucleotides which hybridize to the hereinabovedescribed polynucleotides encode a polypeptide which retains the samebiological functions and activities as the mature polypeptide of SEQ IDNO: 2.

[0056] The invention also relates to nucleic acid fragments hybridizingwith the hereinabove sequence. As used in the present invention, thelength of the “nucleic acid fragment” is at least 10 bp, preferably atleast 20-30 bp, more preferably at least 50-60 bp, and most preferablyat least 100 bp. The nucleic acid fragment can be used in amplificationtechniques of nucleic acid, such as PCR, so as to determine and/orisolate the polynucleotide encoding calcitonin 11.

[0057] The polypeptide and polynucleotide of the invention arepreferably in the isolated form, preferably purified to be homogenous.

[0058] According to the invention, the specific nucleic acid sequenceencoding calcitonin 11 can be obtained in various ways. For example, thepolynucleotide is isolated by hybridization techniques well-known in theart, which include, but are not limited to 1) the hybridization betweena probe and genomic or cDNA library so as to select a homologouspolynucleotide sequence, and 2) antibody screening of expression libraryso as to obtain polynucleotide fragments encoding polypeptides havingcommon structural features.

[0059] According to the invention, DNA fragment sequences may further beobtained by the following methods: 1) isolating double-stranded DNAsequence from genomic DNA; and 2) chemical synthesis of DNA sequence soas to obtain double-stranded DNA.

[0060] Among the above methods, the isolation of genomic DNA is leastfrequently used. A commonly used method is the direct chemical synthesisof DNA sequence. A more frequently used method is the isolation of cDNA.Standard methods for isolating the cDNA of interest is to isolate mRNAfrom donor cells that highly express the gene, followed by reversetranscription of mRNA, and the establishment of plasmid or phage cDNAlibrary. There are many established techniques for extracting mRNA andthe kits are commercially available (e.g. Qiagene). Conventional methodcan be used to construct a cDNA library (Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York,1989). cDNA libraries are also commercially available. For example,Clontech Ltd. has various cDNA libraries. When PCR is further used, evenan extremely small amount of expression products can be cloned.

[0061] Numerous well-known methods can be used for screening for thepolynucleotide of the invention from a cDNA library. These methodsinclude, but are not limited to, (1) DNA-DNA or DNA-RNA hybridization;(2) the appearance or loss of the function of the marker-gene; (3) thedetermination of the level of calcitonin 11 transcripts; (4) thedetermination of protein product of gene expression by immunologymethods or the biological activity assays. The above methods can be usedalone or in combination.

[0062] In method (1), the probe used in the hybridization could behomologous to any portion of polynucleotide of invention. The length ofprobe is typically at least 10 nucleocides, preferably at least 30nucleocides, more preferably at least 50 nucleocides, and still morepreferably at least 100 nucleotides. Furthermore, the length of theprobe is usually less than 2000 nucleotides, preferably less than 1000nucleotides. The probe usually is the DNA sequence chemicallysynthesized on the basis of the sequence information. Of course, thegene of the invention itself or its fragment can be used as a probe.Labels for DNA probes include radioactive isotopes, fluoresceins orenzymes such as alkaline phosphatase.

[0063] In method (4), the detection of the protein products expressed bycalcitonin 11 gene can be carried out by immunology methods, such asWestern blotting, radioimmunoassay, and ELISA.

[0064] The method of amplification of DNA/RNA by PCR (Saiki, et al.Science 1985; 230:1350-1354) is preferably used to obtain thepolynucleotide of the invention. Especially when it is difficult toobtain the full-length cDNA, the method of RACE (Random Amplification ofcDNA Ends) is preferably used. The primers used in PCR can be selectedaccording to the polynucleotide sequence information disclosed herein,and can be synthesized by conventional methods. The amplified DNA/RNAfragments can be isolated and purified by conventional methods such asgel electrophoresis.

[0065] Sequencing of polynucleotide sequence of the gene of theinvention or its various DNA fragments can be carried out by theconventional dideoxy sequencing method (Sanger et al. PNAS, 1977, 74:5463-5467). Sequencing of polynucleotide sequence can also be carriedout using the commercially available sequencing kits. In order to obtainthe full-length cDNA sequence, it is necessary to repeat the sequencingprocess. Sometimes, it is needed to sequence the cDNA of several clonesto obtain the full-length cDNA sequence.

[0066] The invention further relates to a vector comprising thepolynucleotide of the invention, a genetically engineered host celltransformed with the vector of the invention or directly with thesequence encoding calcitonin 11, and a method for producing thepolypeptide of the invention by recombinant techniques.

[0067] In the present invention, the polynucleotide sequences encodingcalcitonin 11 may be inserted into a vector to form a recombinant vectorcontaining the polynucleotide of the invention. The term “vector” refersto a bacterial plasmid, bacteriophage, yeast plasmid, plant virus ormammalian virus such as adenovirus, retrovirus or any other vehicleknown in the art. Vectors suitable for use in the present inventioninclude, but are not limited to the T7-based expression vector forexpression in bacteria (Rosenberg, et al., Gene, 56:125), the pMSXNDexpression vector for expression in mammalian cells (Lee and Nathans, JBiol. Chem., 263:3521, 1988) and baculovirus-derived vectors forexpression in insect cells. Any plasmid or vector can be used toconstruct the recombinant expression vector as long as it can replicateand is stable in the host. One important feature of an expression vectoris that the expression vector typically contains an origin ofreplication, a promoter, a marker gene as well as translation regulatorycomponents.

[0068] Methods known in the art can be used to construct an expressionvector containing the DNA sequence of calcitonin 11 and appropriatetranscription/translation regulatory components. These methods includein vitro recombinant DNA technique, DNA synthesis technique, in vivorecombinant technique and so on (Sambroook, et al. Molecular Cloning, aLaboratory Manual, cold Spring Harbor Laboratory. New York, 1989). TheDNA sequence is operatively linked to a proper promoter in an expressionvector to direct the synthesis of mRNA. Exemplary promoters are lac ortrp promoter of E. coli; PL promoter of A phage; eukaryotic promotersincluding CMV immediate early promoter, HSV thymidine kinase promoter,early and late SV40 promoter, LTRs of retroviruses, and other knownpromoters which control gene expression in the prokaryotic cells,eukaryotic cells or viruses. The expression vector may further comprisea ribosome binding site for initiating translation, transcriptionterminator and the like. Transcription in higher eukaryotes is increasedby inserting an enhancer sequence into the vector. Enhancers arecis-acting elements of DNA, usually about from 10 to 300 bp in lengththat act on a promoter to increase gene transcription level. Examplesinclude the SV40 enhancer on the late side of the replication origin 100to 270 bp, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0069] Further, the expression vector preferably comprises one or moreselective marker genes to provide a phenotype for the selection of thetransformed host cells, e.g., the dehydrofolate reductase, neomycinresistance gene and GFP (green flurencent protein) for eukaryotic cells,as well as tetracycline or ampicillin resistance gene for E. coli.

[0070] An ordinarily skilled in the art knows clearly how to selectappropriate vectors, transcriptional regulatory elements, e.g.,promoters, enhancers, and selective marker genes.

[0071] According to the invention, polynucleotide encoding calcitonin 11or recombinant vector containing the polynucleotide can be transformedor transfected into host cells to construct genetically engineered hostcells containing the polynucleotide or the recombinant vector. The term“host cell” means prokaryote, such as bacteria; or lower eukaryote, suchas yeast; or higher eukaryotic, such as mammalian cells. Representativeexamples are bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; plant cells; insect cells suchas Drosophila S2 or Sf9; animal cells such as CHO, COS or Bowesmelanoma.

[0072] Transformation of a host cell with a DNA sequence of theinvention or a recombinant vector containing the DNA sequence may becarried out by conventional techniques as are well known to thoseskilled in the art. When the host is prokaryotic, such as E. coli,competent cells, which are capable of DNA uptake, can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl2 method using procedures well known in the art.Alternatively, MgCl₂ can be used. Transformation can also be carried outby electroporation, if desired. When the host is an eukaryote,transfection methods as well as calcium phosphate precipitation may beused. Conventional mechanical procedures such as micro-injection,electroporation, or liposome-mediated transfection may also be used.

[0073] The recombinant calcitonin 11 can be expressed or produced by theconventional recombinant DNA technology (Science, 1984; 224:1431), usingpolynucleotide sequence of the invention. The steps generally include:

[0074] (1) transfecting or transforming the appropriate host cells withthe polynucleotide (or variant) encoding calcitonin 11 of the inventionor the recombinant expression vector containing the polynucleotide;

[0075] (2) culturing the host cells in an appropriate medium; and

[0076] (3) isolating or purifying the protein from the medium or cells.

[0077] In Step (2) above, depending on the host cells used, the mediumfor cultivation can be selected from various conventional mediums. Thehost cells are cultured under a condition suitable for its growth untilthe host cells grow to an appropriate cell density. Then, the selectedpromoter is induced by appropriate means (e.g., temperature shift orchemical induction) and cells are cultured for an additional period.

[0078] In Step (3), the recombinant polypeptide may be included in thecells, or expressed on the cell membrane, or secreted out of the cell.If desired, physical, chemical and other properties can be utilized invarious isolation methods to isolate and purify the recombinant protein.These methods are well-known to those skilled in the art and include,but are not limited to conventional renaturation treatment, treatment bya protein precipitant (such as salt precipitation), centrifugation, celllysis by osmosis, sonication, supercentrifugation, molecular sievechromatography or gel chromatography, adsorption chromatography, ionexchange chromatography, HPLC, and any other liquid chromatography, anda combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The following drawings are provided to illustrate the embodimentof the invention, not to limit the scope of invention defined by theclaims.

[0080]FIG. 1 is the homology comparison of the amino acid sequence ofcalcitonin 11 in this invention with the amino acid sequence of domaincalcitonin family proteins in amino acid sequence from No. 12 to No. 95.“|”, “:” and “.” shows that the probability of appearance of differentamino acid in the same position in two sequence is reduced in turn.

[0081]FIG. 2 shows an SDS-PAGE of the isolated calcitonin 11, which hasa molecular weight of 11 kDa. The isolated protein band is marked withan arrow.

EXAMPLES

[0082] The invention is further illustrated by the following examples.It is appreciated that these examples are only intended to illustratethe invention, not to limit the scope of the invention. For theexperimental methods in the following examples, they are performed underroutine conditions, e.g., those described by Sambrook. et al., inMolecule Clone: A Laboratory Manual, New York: Cold Spring HarborLaboratory Press, 1989, or as instructed by the manufacturers, unlessotherwise specified.

Example 1

[0083] Cloning of Calcitonin 11 Gene

[0084] Total RNA from a human embryonic brain was extracted by theone-step method with guanidinium isocyanate/phenol/chloroform. The poly(A) mRNA was isolated from the total RNA with Quik mRNA Isolation Kit(Qiegene). cDNA was prepared by reverse transcription with 2 μg poly (A)mRNA. The cDNA fragments were inserted into the polyclonal site of PBSK(+) vector (Clontech) using Smart cDNA cloning kit (Clontech) and thentransformed into DH5α to form the cDNA library. The 5′- and 3′-ends ofall clones were sequenced with Dye terminate cycle reaction sequencingkit (Perkin-Elmer) and ABI 377 Automatic Sequencer (Perkin-Elmer). Thesequenced cDNA were compared with the public database of DNA sequences(Genebank) and the DNA sequence of one clone 0259f12 was found to be anovel DNA sequence. The inserted cDNA sequence of clone 0259f12 wasdual-directionally sequenced with a serial of synthesized primers. Itwas indicated that the full length cDNA contained in clone 0259f12 was1614 bp (SEQ ID NO: 1) with a 291 bp ORF located in positions 211-501which encodes a novel protein (SEQ ID NO: 2). This clone was namedpBS-0259f12 and the encoded protein was named calcitonin 11.

Example 2

[0085] Domain Analysis of cDNA Clone

[0086] The DNA sequence of the calcitonin 11 gene and the amino acidsequence it encoded was analyzed with the profile scan program in GCG(Basic Local Alignment Search Tool) (Altschul, et al., J. Mol. Biol.,1990; 215:403-10), and domain analysis was carried out in databanks,such as Prosite. We found that calcitonin 11 is homologous to thecharacteristic motif of the calcitonin family proteins at the amino acidlevel, between positions No. 12 and No. 95 (probability is 0.2, thescore is 11.36; threshold value is 11.32).

Example 3

[0087] Cloning Calcitonin 11 Gene by RT-PCR

[0088] Using total RNA extracted from a human embryonic brain astemplate, reverse transcription was carried out with oligo-dT primer toproduce cDNAs. The cDNA was purified with a Qiagen Kit, and PCR wascarried out with the following primers: Primer 1:5′-AATTATTTTCCTGCAGTGGTTTGC-3′ (SEQ ID NO:3) Primer 2:5′-GTGCTTTAACGAAGTTGTTTTATC-3′ (SEQ ID NO:4)

[0089] Primer 1 is the forward sequence started from position 1 of 5′end of SEQ ID NO: 1.

[0090] Primer 2 is the reverse sequence of the 3′ end of SEQ ID NO: 1.

[0091] The amplification condition was a 50 μl reaction systemcontaining 50 mmol/L KCl, 10 mmol/L Tris-Cl (pH8.5), 1.5 mmol/L MgCl₂,200 μmol/L dNTP, 10 pmol of each primer, 1U Taq DNA polymerase(Clontech). The reaction was performed on a PE 9600 DNA amplifier withthe following parameters: 94° C. 30 sec, 55° C. 30 sec, and 72° C. 2 minfor 25 cycles. β-actin was used as a positive control, and a blanktemplate, as a negative control in RT-PCR. The amplified products werepurified with a QIAGEN kit, and linked with a pCR vector (Invitrogen)using a TA Cloning Kit. DNA sequencing results show that the DNAsequence of PCR products was identical to nucleotides 1-1614 bp of SEQID NO: 1.

Example 4

[0092] Northern Blotting of Expression of Calcitonin 11 Gene

[0093] Total RNA was extracted by one-step method (Anal. Biochem., 1987,162, 156-159) with guanidinium isocyanate-phenol-chloroform. That is,homogenize the tissue using 4M guanidinium isocyanate-25mM sodiumcitrate and 0.2M sodium acetate (pH 4.0), add 1 volume phenol and ⅕volume chloroform-isoamyl alcohol (49:1), centrifuge after mixing. Takethe acqueous phase, add 0.8 volume isopropyl alcohol, then centrifugethe mixture. Wash the RNA precipitation using 70% ethanol, dry, anddissolve it in water. 20 μg RNA was electrophoresed on the 1.2% agarosegel containing 20 mM 3-(N-morpholino) propane sulfonic acid (pH 7.0)-5mM sodium acetate-1 mM EDTA-2.2 M formaldehyde. Then transfer it to anitrocellulose filter. Prepare the ³²P-labelled DNA probe with α-³²PdATP by the random primer method. The used DNA probe is the codingsequence (211 bp-501 bp) of calcitonin 11 amplified by PCR as indicatedin FIG. 1. The nitrocellulose filter with the transferred RNA washybridized with the ³²P-labelled DNA probe (2×10⁶ cpm/ml) overnight in abuffer containing 50% formamide-25 mM KH₂PO₄ (Ph7.4)-5×Denhardt'ssolution and 200 μg/ml salmine and washed in 1×SSC-0.1% SDS, at 55° C.,for 30 min. The filter was then analyzed and quantified using a phosphorimager.

Example 5

[0094] In Vitro Expression, Isolation and Purification of RecombinantCalcitonin 11

[0095] A pair of primers for specific amplification was designed basedon SEQ ID NO: 1 and the encoding region in FIG. 1, the sequences are asfollows: (SEQ ID No:5) Primer 3:5′-CCCCATATGGATTGATGTAGAGATTGGCCAATGA-3′ (SEQ ID No:6) Primer 4:5′-CATGGATCCTCACAAAGTTGCTGAGCCCACACAT-3′

[0096] These two primers contain a NdeI and BamHI cleavage site on the5′ end respectively. Within the sites are the coding sequences of the 5′and 3′ end of the desired gene. NdeI and BamHI cleavage sites werecorresponding to the selective cleavage sites on the expression vectorpET-28b (+) (Novagen, Cat. No. 69865.3). PCR amplification was performedwith the plasmid pBS-0259f12 containing the full-length target gene as atemplate. The PCR reaction was performed in a total volume of 50 μlcontaining 10 pg pBS-0259f12 plasmid, 10 pmol of Primer-3 and 10 pmol ofPrimer-4, and 1 μl of Advantage polymerase Mix (Clontech). Theparameters of PCR were 94° C. 20 sec, 60° C. 30 sec, and 68° C. 2 minfor 25 cycles. After digesting the amplification products and theplasmid pET-28 (+) by NdeI and BamHI, the large fragments were recoveredand ligated with T4 ligase. The ligated product was transformed into E.coli DH5α cells with the calcium chloride method. After culturedovernight on an LB plate containing a final concentration of 30 μg/mlkanamycin, positive clones were selected using colony PCR and thensequenced. The positive clone (pET-0259f12) with the correct sequencewas selected out and the recombinant plasmid thereof was transformedinto BL21 (DE3)plySs (Novagen) using the calcium chloride method. In anLB liquid medium containing a final concentration of 30 μg/ml ofkanamycin, the host bacteria BL21 (pET-0259f12) were cultured at 37° C.to the exponential growth phase, then IPTG were added with the finalconcentration of 1 mmol/L. The cells were cultured for another 5 hours,and then centrifuged to harvest the bacteria. After the bacteria weresonicated, the supernatant was collected by centrifugation. Then thepurified desired protein-calcitonin 11 was obtained by a His.Bind QuickCartridge (Novagen) affinity column with binding 6His-Tag. SDS-PAGEshowed a single band at 11 kDa (FIG. 2). The band was transferred ontothe PVDF membrane and the N-terminal amino acid was sequenced by EdamsHydrolysis, which shows that the first 15 amino acids on N-terminus wereidentical to those in SEQ ID NO: 2.

Example 6

[0097] Preparation of Antibody Against Calcitonin 11

[0098] The following specific calcitonin 11 polypeptide was synthesizedby a polypeptide synthesizer (PE-ABI):NH₂-Met-Ser-Leu-Ala-Asn-Leu-Tyr-IIe-Asn-His-Trp-Gln-Gly-Glu-Leu-COOH(SEQ ID NO:7). The polypeptide was conjugated with hemocyanin and bovineserum albumin (BSA) respectively to form two composites (See Avrameas etal., Immunochemistry, 1969, 6:43). 4 mg of hemocyanin-polypeptidecomposite was used to immunize rabbit together with Freund's completeadjuvant. The rabbit was re-immunized with the hemocyanin-polypeptidecomposite and Freund's incomplete adjuvent 15 days later. The titer ofantibody in the rabbit sera was determined with a titration plate coatedwith 15 μg/ml BSA-polypeptide composite by ELISA. Total IgG was isolatedfrom the sera of an antibody positive rabbit with Protein A-Sepharose.The polypeptide was bound to Sepharose 4B column activated by cyanogenbromide. The antibodies against the polypeptide were isolated from thetotal IgG by affinity chromatography. Immunoprecipitation confirmed thatthe purified antibodies could specifically bind to calcitonin 11.

Example 7

[0099] Application of the Polynucleotide Fragments of the Invention asHybridization Probes

[0100] Oligonucleotides probes selected from the polynucleotide of theinvention have many applications. The probe could be used to determinethe existence of polynucleotide of the invention or its homologouspolynucleotide sequences by hybridization with a genomic or cDNA libraryof normal or clinical tissues from various sources. The probes could befurther used to determine whether polynucleotide of the invention or itshomologous polynucleotide sequences are abnormally expressed in cellsfrom normal or clinical tissues.

[0101] The purpose of the following example is to select suitableoligonucletide fragments from SEQ ID NO: 1 as hybird probes to apply inmembrane hybridization to determine whether there is any polynucleotideof the invention or its homologous polynucleotide sequences in sampletissues. Membrane hybridization methods include dot blot, Southern blot,Northern blot, and replica hybridization. All methods follow nearly thesame steps after the polynucleotide samples are immobilized onmembranes. These steps are: membranes with immobilized samples areprehybridized in hybridization buffer not containing probes to blocknonspecific binding sites of the membranes. Then the prehybridizationbuffer is replaced by hybridization buffer containing labeled probes andincubation is carried out at an appropriate temperature so probeshybridize with the target nucleotides. Free probes are washed off by aseries of washing steps after the hybridization step. A high-stringencywashing condition (relatively low salt concentration and hightemperature) is applied in the example to reduce background and retainhighly specific signals. Two types of probes are selected for theexample: the first type of probes are oligonucleotides identical orannealed to SEQ ID NO:1; the second type probes are oligonucleotidespartially identical or partially annealed to SEQ ID NO:1. Dot blotmethod is applied in the example for immobilization of the samples onmembrane. The strongest specific signal produced by hybridizationbetween first type probes and samples is obtained after relativelystringent membrane washing steps.

[0102] Selection of Probes

[0103] The principles below should be followed for the selection ofoligonucleotide fragments from SEQ ID NO:1 as hybrid probes:

[0104] 1. The optimal length of probes should be between eighteen andfifty nucleotides.

[0105] 2. GC content should be between 30% and 70%, since nonspecifichybridization increases when GC content is more than 70%.

[0106] 3. There should be no complementary regions within the probesthemselves.

[0107] 4. Probes meeting the requirements above could be initiallyselected for further computer-aided sequence analysis, which includeshomology comparison between the initially selected probes and its sourcesequence region (SEQ ID NO: 1), other known genomic sequences and theircomplements. Generally, the initial selected probes should not be usedwhen they share fifteen identical continuous base pairs, or 85% homologywith non-target region.

[0108] 5. Whether the initially selected probes should be chosen forfinal application depends upon further experimental confirmation.

[0109] The following two probes could be selected and synthesized afterthe analysis above:

[0110] Probe One belongs to the first type, which is completelyidentical or annealed to the gene fragments of SEQ ID NO: 1 (41nucleotides):

5′-GGCAAGGTGAACTGGAATCTCAGCGATTGGCTACAGCAGTT-3′  (SEQ ID NO: 8)

[0111] Probe Two belongs to the second type which is a substituted ormutant sequence of a fragment of SEQ ID NO: 1 (41 nucleotides):

5′-GGATCGGTGAACTGGAAATCGAGCGATTGGCTAATCGAGTT-3′  (SEQ ID NO: 9)

[0112] Other frequently used reagents not listed but involved in thefollowing experimental steps and their preparation methods can be found,for example, in: DNA PROBES G. H. Keller; M. M. Manak; Stockton Press,1989 (USA) or a more commonly used molecular cloning experimentalhandbook Molecular Cloning (J. Sambrook et al., Academic Press, 1998,2nd Edition).

[0113] Sample Preparation:

[0114] 1. DNA Extraction from Fresh or Frozen Tissues

[0115] Steps: 1) Place fresh or newly thawed tissue onto a dish on icecontaining phosphate-buffered saline (PBS). Cut the tissue into smallpieces with scissors or an operating knife. Tissues should be kept dampthrough the operation. 2) Mince the tissue by centrifugation at 2,000 gfor 10 minutes. 3) Re-suspend the pellet (about 10 ml/g) with coldhomogenating buffer (0.25 mol/l saccharose; 25 mmol/l Tris-HCl, pH7.5;25 m mol/LnaCl; 25 mmol/L MgCl2) at 4° C., and homogenize the tissuesuspension at full speed with an electronic homogenizer. 5) Centrifugeat 1,000 g for 10 minutes. 6) Re-suspend the cell pellet (1-5 ml per 0.1g initial tissue sample), and centrifuge at 1,000 g for 10 minutes. 7)Re-suspend the pellet with lysis buffer (1-5 ml per 0.1 g initial tissuesample), and continue on to the phenol extraction step described below.

[0116] 2. Phenol Extraction of DNA

[0117] Steps: 1) Wash cells with 1-10 ml cold PBS buffer and centrifugeat 1000 g for 10 minutes. 2) Re-suspend the precipitated cells with atleast 100 μl cold cell lysis buffer (1×108 cells/ml). 3) Add SDS to afinal concentration of 1%. Addition of SDS into the cell precipitationbefore cell re-suspension will cause the formation of large cellaggregates difficult to homogenize and reduce total yield. This isespecially important when more than 10⁷ cells are used. 4) Incubate at50° C. for an hour or shake gently overnight at 37° C. 5) Add an equalvolume of phenol: chloroform: isoamyl alcohol (25:24:1) to the DNAsolution to be purified in a microcentrifuge tube, and centrifuge for 10minutes. If the two phases are not clearly separated, the solutionshould be recentrifuged. 6) Remove the acqueous phase to a new tube. 7)Add an equal volume of chloroform: isoamyl alcohol (24:1) and centrifugefor 10 minutes. 8) Remove the water phase containing DNA to a new tubeand then purify DNA by ethanol precipitation.

[0118] 3. DNA Purification by Ethanol Precipitation

[0119] Steps: 1) Add 1/10 vol of 2 mol/L sodium acetate and 2 vol ofcold 100% ethanol into the DNA solution, mix and place at −20° C. for anhour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully remove theethanol. 4) Add 500 μl of cold 70% ethanol to wash the pellet andcentrifuge for 5 minutes. 6) Carefully remove the ethanol and invert thetube on absorbent paper to remove remnant ethanol. Air dry for 10-15minutes to evaporate the ethanol on the pellet surface. Do not dry thepellet completely since completely dry pellet is difficult to bedissolved again. 7) Re-suspend the DNA pellet with a small volume of TEor water. Spin at low speed or blow with a pipette, and add TE graduallyand mix until DNA is completely dissolved. About 1 μl of DNA solution isobtained per 1˜5×10⁶ cells.

[0120] The following steps 8-13 are applied only when contamination mustbe removed, otherwise go directly to step 14. 8) Add RNase A into DNAsolution to a final concentration of 100 μg/ml and incubate at 37° C.for 30 minutes. 9) Add SDS and protease K to the final concentration of0.5% and 100 μg/ml individually, and incubate at 37° C. for 30 minutes.10) Add an equal volume of phenol: chloroform: isoamyl alcohol(25:24:1), and centrifuge for 10 minutes. 11) Carefully remove the waterphase and extract it with an equal volume of chloroform: isoamyl alcohol(24:1) and centrifuge for 10 minutes. 12) Carefully remove the waterphase, and add 1/10 vol of 2 mol/L sodium acetate and 2.5 vol of cold100% ethanol, then mix and place at −20° C. for an hour. 13) Wash thepellet with 70% ethanol and 100% ethanol, air dry and re-suspend DNA assame as the steps 3-6. 14) Determine the purity and production of DNA byA260 and A280 assay. 15) Separate DNA sample into several portions andstore at −20° C.

[0121] Preparation of Sample Membrane

[0122] 1) Take 4×2 pieces of nitrocellulose membrane (NC membrane) ofdesired size, and lightly mark out the sample dot sites and samplenumber with a pencil. Every probe needs two pieces of NC membrane, sothen membranes could be washed under high stringency condition andmoderate stringency condition individually in the following experimentalsteps.

[0123] 2) Pipette 15 μl of samples and control individually, dot them onthe membrane, and dry at room tempreture.

[0124] 3) Place the membranes on filter paper soaked in 0.1 mol/LnaOH,1.5 mol/L NaCl, leave for 5 minutes (twice), and allow to dry. Transferthe membranes on filter paper soaked in 0.5 mol/L Tris-HCl (pH7.0), 3mol/L NaCl, leave for 5 minutes (twice), and allow to dry.

[0125] 4) place the membranes between clean filter paper, packet withaluminum foil, and vacuum dry at 60-80° C. for 2 hours.

[0126] Labeling of Probes

[0127] 1) Add 3 μl probe (0.1 OD/10 μl), 2 μl kinase buffer, 8-10 uCiγ-³²P-dATP+2U Kinase, and add water to the final volume of 20 μl.

[0128] 2) Incubate at 37° C. for 2 hours.

[0129] 3) Add 1/5 vol bromophenol blue indicator (BPB).

[0130] 4) Load that sample on Sephadex G-50 column.

[0131] 5) Collect the first peak before the elution of ³²P-Probe(monitor the eluting process by Monitor).

[0132] 6) Five drops each tube and collect for 10-15 tubes.

[0133] 7) Measure the isotope amount with liquid scintillator.

[0134] 8) Merged collection of the first peak is the prepared ³²P-Probe(the second peak is free γ-³²P-dATP).

[0135] Prehybridization

[0136] Place the sample membranes in a plastic bag, add 3-10 mgprehybrid buffer (10×Denhardt's; 6×SSC, 0.1 mg/ml CT DNA (calf thymusgland DNA)), seal the bag, and shake on a 68° C. water bath for twohours hybridization.

[0137] Cut off a corner of the plastic bag, add in prepared probes, sealthe bag, and shake on a 42° C. water bath overnight.

[0138] Membrane Washing

[0139] Membrane washing applying a high-stringency condition:

[0140] 1) Take out the hybridized sample membranes

[0141] 2) Wash the membranes with 2×SSC, 0.1% SDS at 40° C. for 15minutes (twice).

[0142] 3) Wash the membranes with 0.1×SSC, 0.1% SDS at 40° C. for 15minutes (twice).

[0143] 4) Wash the membranes with 0.1×SSC, 0.1% SDS at 55° C. for 30minutes (twice), and dry at room temperature.

[0144] Membrane washing applying a low-stringency condition:

[0145] 1) Take out the hybridized sample membranes.

[0146] 2) Wash the membranes with 2×SSC, 0.1% SDS at 37° C. for 15minutes (twice).

[0147] 3) Wash the membranes with 0.1×SSC, 0.1% SDS at 37° C. for 15minutes (twice).

[0148] 4) Wash the membranes with 0.1×SSC, 0.1% SDS at 40° C. for 15minutes (twice), and dry at room temperature.

[0149] X Ray Autoradiography:

[0150] X ray autoradiograph at −70° C. (autoradiograph time variesaccording to radioactivity of the hybrid spots).

[0151] Experimental Results:

[0152] In hybridization experiments carried out under low-stringencymembrane washing condition, the radioactivity of all the above twoprobes hybridization spots show no obvious difference; while inhybridization experiments carried out under high-stringency membranewashing condition, radioactivity of the hybrid spot by probe one isobviously stronger than that of the other probes. So Probe One could beapplied in qualitative and quantitative analyses of the existence anddifferential expression of the polynucleotide of the invention indifferent tissues.

Example 8

[0153] DNA Microarray

[0154] DNA-chip or DNA Microarray technology is now studied anddeveloped in many laboratories and large pharmaceutical companies. Thetechnology uses a large number of target genes that are arrayed on aglass or silicon slide with high density, then uses fluorescencedetection and software to compare and analyze the data, so it cananalyze a large amount scale of biology information quickly andeffectively. The polynucleotide provided in this invention may be usedas target DNA by DNA-chip technology to find new gene function, screentissue-specific genes especially tumor related gene, and for disease(hereditary diseases etc.) diagnosis. The methods are known in the art(see e.g., DeRisi, et al. 1997, Science 278, 680-686; Helle, et al.1997, PNAS 94:2150-2155.)

[0155] 1. DNA Fixation

[0156] 4000 cDNAs of various lengths were used as target DNA, includingthe polyneucleotide of this invention. cDNAs were amplified by PCR andpurified, then adjusted to about 500 ng/μl. PCR products were printed onglass slide using Cartesian 7500 Robotics (Cartesian, USA), with a gapof 280 μm.

[0157] Printed arrays were hydrated, dried, UV cross-linked, rinsed anddried to fix the DNA on the glass slide. The details of the method havebeen reported in many documents. The step after fixation in this exampleis:

[0158] 1) Incubate for 4 hr in a humid chamber;

[0159] 2) Wash 1 min in 0.2% SDS;

[0160] 3) Wash 2×1 min in ddH₂O;

[0161] 4) Block for 5 min with NaBH₄;

[0162] 5) Incubate in water at 95° C. for 2min;

[0163] 6) Wash 1 min in 0.2% SDS;

[0164] 7) Wash twice with ddH₂0;

[0165] 8) Air dry and store in the dark at 25° C.

[0166] 2. Probe labeling

[0167] Total mRNA was extracted from normal liver cells and liver cancercells by the one-step method, then purified using Oligotex mRNA Midi Kit(Qiagen). Following the reverse transcriptase step, mRNA from normalliver cells was labeled with Cy3dUTP (5-Amino-propargyl-2,-deoxyuridine5,-triphate coupled to the Cy3 fluorescent dye, purchased from AmershamPhamacia Biotech Co.) and mRNA from liver cancer cells was labeled withCy5dUTP (5-Amino-propargyl-2,-deoxyuridine 5,-triphate coupled to Cy5fluorescent dye, purchased from Amersham Phamacia Biotech Co.). Thenlabeled probes were purified. (See Schena, et al., 1996, Proc.Natl.Acad.Sci., USA. 93:10614-10619; Schena, et al., 1995, Science.270:467-480.).

[0168] 3. Hybridization

[0169] Labeled probes from each tissue were mixed with the DNA-chip inUniHyb™ Hybridization Solution (TeleChem) for 16 hr. After washing withwashing buffer (1×SSC, 0.2% SDS) at room temperature, Arrays were scanedusing ScanArray 3000 (General Scanning Co., USA). The images wereanalyzed with Imagene (Biodiscovery Co., USA). The ratios of Cy3 to Cy5were obtained. If the ratio is less than 0.5 or larger than 2, we canconclude the gene was expressed differently in two tissues.

[0170] The results showed that Cy3 signal=2005.25 ( average of fourexperiments), Cy5 signal=1905.46 (average of four experiments),Cy3/Cy5=1.0281. So there is no obvious differential expression in thetwo tissues of the polynucleotide provided in this invention.

Industrial Applicability

[0171] The polypeptide of the invention and antagonists, agonists andinhibitors thereof can be directly used for the treatment of diseases,e.g., various malignant tumors or cancers, dermatitis, inflammation,adrenoprival disease and HIV infection and immune system diseases.

[0172] Calcitonin, CGRP and IAPP have a similar structural motif.Calcitonin is very important in the regulation of calcium concentrationin the blood. The regulatory functions of calcitonin are closely relatedto physiological functions of parathormone, gastrin, pancreatin andglucagon. CGRP can cause vasodilatation in different vessels (includingcoronary artery, blood vessel of brain, and the entire vascular system),and it is an important factor in regulating blood flow and blood vesseltension. Furthermore, CGRP is also important in the modulation of somefunctions of nervous system, cellular behavior and endocrine systemfunctions. IAPP can selectively inhibit glucose metabolism and glycogensynthesis induced by insulin.

[0173] Abnormal expression of calcitonin, CGRP, IAPP and the polypeptideof this invention may cause abnormal blood calcium level, cardiovascularsystem disorders, and abnormal blood sugar levels. These may furthercause many diseases, for example: infant hyperglycemia, adulthypercalcimia, rachitis, osteomalacia, myeloma, parathyroid cyst,parathyrodioma, coronary heart disease, hypertension, diabetes, anddiabetes and relted syndromes, aw well as certain hereditary diseases,hemopathy and immune diseases.

[0174] The polypeptide of the invention and its antagonist, agonist andinhibitors can be directly applied to treat some diseases, for example:infant hyperglycemia, adult hypercalcinemia, rachitis, osteomalacia,myeloma, parathyroid cyst, parathyrodioma, coronary heart disease,hypertension, diabetes, diease interrelated with diabetes, somehereditarydisease, hemopathy and immune diseases, etc.

[0175] The invention also provides methods for screening compounds so asto identify an agent which enhances calcitonin 11 activity (agonists) ordecrease calcitonin 11 activity (antagonists). The agonists enhance thebiological functions of calcitonin 11 such as inactivation of cellproliferation, while the antagonists prevent and alleviate disordersassociated with excessive cell proliferation, such as various cancers.For example, in the presence of an agent, mammalian cells or membranepreparations expressing calcitonin 11 can be incubated with a labeledcalcitonin 11 to determine the ability of the agent to enhance orrepress the interaction.

[0176] Antagonists of calcitonin 11 include antibodies, compounds,receptor deletants and analogues. Antagonists of calcitonin 11 can bindto calcitonin 11 and eliminate or reduce its activity or inhibit theproduction, or bind to the active site of the polypeptide so that thepolypeptide can not function biologically.

[0177] When screening for compounds as antagonists, calcitonin 11 may beadded to a biological assay. It can be determined whether the compoundis an antagonist or not by determining its effect on the interactionbetween calcitonin 11 and its receptor. Using the same method as thatfor screening compounds, receptor deletants and analogues acting asantagonists can be selected. Polypeptide molecules capable of binding tocalcitonin 11 can be obtained by screening a polypeptide librarycomprising various combinations of amino acids bound onto a solidmatrix. Calcitonin 11 is preferably labeled in the screening.

[0178] The invention further provides a method for producing antibodiesusing the polypeptide, fragments, derivatives, or analogues thereof, orcells comprising the polypeptide as an antigen. These antibodies may bepolyclonal or monoclonal antibodies. The invention also providesantibodies against epitopes of calcitonin 11. These antibodies include,but are not limited to, polyclonal antibody, monoclonal antibody,chimeric antibody, single-chain antibody, Fab fragment and the fragmentsproduced by a Fab expression library.

[0179] Polyclonal antibodies can be prepared by immunizing animals, suchas rabbit, mouse, and rat, with calcitonin 11. Various adjuvants,including but are not limited to Freund's adjuvant, can be used toenhance the immunization. The techniques for producing calcitonin 11monoclonal antibodies include, but are not limited to, the hybridomatechnique (Kohler and Milstein, 1975, Nature 256:495-497), the triomatechnique, the human B-cell hybridoma technique, the EBV-hybridomatechnique and so on. A chimeric antibody comprising a constant region ofhuman origin and a variable region of non-human origin can be producedusing methods well-known in the art (Morrison et al, 1985, PNAS81:6851). Furthermore, techniques for producing a single-chain antibody(U.S. Pat. No. 4,946,778) are also useful for preparing single-chainantibodies against calcitonin 11.

[0180] The antibody against calcitonin 11 can be used inimmunohistochemical method to detect the presence of calcitonin 11 in abiopsy specimen.

[0181] The monoclonal antibody specific to calcitonin 11 can be labeledby radioactive isotopes, and used in vivo to trace the location anddistribution of calcitonin 11. This radioactively labeled antibody canbe used in a non-invasive diagnostic method for the determination oftumor location and metastasis.

[0182] Antibodies can also be designed as an immunotoxin targeting aparticular site in the body. For example, a monoclonal antibody havinghigh affinity to calcitonin 11 can be covalently bound to bacterial orplant toxins, such as diphtheria toxin, ricin, ormosine. One commonmethod is to attack the amino group on the antibody with sulfydrylcross-linking agents, such as SPDP, and bind the toxin onto the antibodyby interchanging the disulfide bonds. This hybrid antibody can be usedto kill calcitonin 11-positive cells.

[0183] The antibody of the invention is useful for the therapy or theprophylaxis of disorders related to the calcitonin 11. The appropriateamount of antibody can be administered to stimulate or block theproduction or activity of calcitonin 11.

[0184] The invention further provides diagnostic assays for quantitativeand in situ measurement of calcitonin 11 level. Methods that can be usedfor these assays are well known in the art and include FISH assay andradioimmunoassay. The level of calcitonin 11 detected in the assay canbe used to illustrate the role of calcitonin 11 in diseases and todetermine diseases associated with calcitonin 11.

[0185] The polypeptide of the invention is useful in the analysis ofpolypeptide profile. For example, the polypeptide can be specificallydigested by physical, chemical, or enzymatic means, and then analyzed byone, two or three dimensional gel electrophoresis, preferably byspectrometry.

[0186] New calcitonin 11 polynucleotides also have many therapeuticapplications. Gene therapy technology can be used in the treatment ofabnormal cell proliferation, development or metabolism, which are causedby the loss of calcitonin 11 expression or the abnormal or non-activeexpression of calcitonin 11. Recombinant gene therapy vectors, such asviral vectors, can be designed to express mutated calcitonin 11 so as toinhibit the activity of endogenous calcitonin 11. For example, one formof mutated calcitonin 11 is a truncated calcitonin 11 whose signaltransduction domain is deleted. Therefore, this mutated calcitonin 11can bind to the downstream substrate without the activity of signaltransduction. Thus, the recombinant gene therapy vectors can be used tocure diseases caused by abnormal expression or activity of calcitonin11. The expression vectors derived from a virus, such as retrovirus,adenovirus, adeno-associated virus, herpes simplex virus, parvovirus,and so on, can be used to introduce the calcitonin 11 gene into thecells. The methods for constructing a recombinant virus vector harboringcalcitonin 11 gene are described in the literature (Sambrook, et al.supra). In addition, the recombinant calcitonin 11 gene can be packedinto liposome and then transferred into the cells.

[0187] The methods for introducing the polynucleotides into tissues orcells include directly injecting the polynucleotides into tissue in thebody; or introducing the polynucleotides into cells in vitro withvectors, such as virus, phage, or plasmid, etc, and then transplantingthe cells into the body.

[0188] Also included in the invention are ribozymes andoligonucleotides, including antisense RNA and DNA, which inhibit thetranslation of the calcitonin 11 mRNA. Ribozyme is an enzyme-like RNAmolecule capable of specifically digesting certain RNA molecules. Themechanism is nucleic acid endo-cleavage following specific hybridizationof ribozyme molecule and the complementary target RNA. Antisense RNA andDNA as well as ribozyme can be prepared by using any conventionaltechniques for RNA and DNA synthesis, e.g., the widely used solid phasephosphite chemical method for oligonucleotide synthesis. Antisense RNAmolecule can be obtained by the in vivo or in vitro transcription of theDNA sequence encoding the RNA, wherein the DNA sequence is integratedinto the vector and downstream of the RNA polymerase promoter. In orderto increase its stability, a nucleic acid molecule can be modified inmany manners, e.g., increasing the length of the two flanking sequences,and replacing the phosphodiester bond with the phosphothioester bond inthe oligonucleotide.

[0189] Polynucleotides encoding calcitonin 11 can be used in thediagnosis of calcitonin 11 related diseases. Polynucleotides encodingcalcitonin 11 can be used to detect whether calcitonin 11 is expressedor not, and whether the expression of calcitonin 11 is normal orabnormal in the case of diseases. For example, calcitonin 11 DNAsequences can be used in the hybridization with biopsy samples todetermine the expression of calcitonin 11. The hybridization methodsinclude Southern blotting, Northern blotting and in situ blotting, etc.,which are well-known and established techniques. The corresponding kitsare commercially available. A part of or all of the polynucleotides ofthe invention can be used as probe and fixed on a microarray or DNA chipfor analysis of differential expression of genes in tissues and for thediagnosis of genes. The calcitonin 11 specific primers can be used inRNA-polymerase chain reaction and in vitro amplification to detecttranscripts of calcitonin 11.

[0190] Further, detection of mutations in calcitonin 11 gene is usefulfor the diagnosis of calcitonin 11 -related diseases. Mutations ofcalcitonin 11 include site mutation, translocation, deletion,rearrangement and any other mutations compared with the wild-typecalcitonin 11 DNA sequence. The conventional methods, such as Southernblotting, DNA sequencing, PCR and in situ blotting, can be used todetect a mutation. Moreover, mutations sometimes affect the expressionof protein. Therefore, Northern blotting and Western blotting can beused to indirectly determine whether the gene is mutated or not.

[0191] Polypeptides of the present invention are also valuable forchromosome identification. The polypeptides can hybridize with aparticular location on an individual human chromosome. There is acurrent need for identifying particular sites of gene on the chromosome.Few chromosomal markers based on actual sequence data (repeatpolymorphism) are presently available for marking chromosomal location.The mapping of DNA to chromosomes according to the present invention isan important first step in correlating those sequences with genesassociated with the disease.

[0192] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-35 bp) from the cDNA. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the primer will yield an amplified fragment.

[0193] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using theoligonucleotide primers of the invention, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in a similar manner. Other chromosome mappingstrategies that can similarly be used to map to its chromosome includein situ hybridization, prescreening with labeled flow-sorted chromosomesand preselection by hybridization to construct chromosome specific-cDNAlibraries.

[0194] A cDNA clone can be precisely mapped to a metaphase chromosomalspread in one step using fluorescence in situ hybridization (FISH). Fora review of this technique, see Verma et al., Human Chromosomes: aManual of Basic Techniques, Pergamon Press, New York (1988).

[0195] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis.

[0196] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be the causeof the disease. Comparison of affected and unaffected individualsgenerally involves first looking for structural alterations in thechromosomes, such as deletions or translocations, that are visible atthe chromosome level, or detectable using PCR based on that DNAsequence. With current resolution of physical mapping and geneticmapping techniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50 to 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0197] According to the invention, the polypeptides, polynucleotides andits mimetics, agonists, antagonists and inhibitors may be employed incombination with a suitable pharmaceutical carrier. Such a carrierincludes but is not limited to water, glucose, ethanol, salt, buffer,glycerol, and combinations thereof. Such compositions comprise a safeand effective amount of the polypeptide or antagonist, as well as apharmaceutically acceptable carrier or excipient with no influence onthe effectiveness of the drug. These compositions can be used as drugsin disease treatment.

[0198] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention. Withsuch container (s) there may be a notice from a governmental agency,that regulates the manufacture, use or sale of pharmaceuticals orbiological products, the notice reflects government's approval for themanufacture, use or sale for human administration. In addition, thepolypeptides of the invention may be employed in conjunction with othertherapeutic compounds.

[0199] The pharmaceutical compositions may be administered in aconvenient manner, such as through topical, intravenous,intraperitoneal, intramuscular, subcutaneous, intranasal or intradermalroutes. Calcitonin 11 is administered in an amount, which is effectivefor treating and/or prophylaxis of the specific indication. The amountof calcitonin 11 administrated to a patient will depend upon variousfactors, such as delivery methods, the subject's health, and thejudgment of the skilled clinician.

1 9 1 1614 DNA Homo sapiens CDS (211)..(501) 1 aattattttc ctgcagtggtttgcaatccc tgtgcttccg ttggcctttc tttccttctt 60 ccaagatgtc tgccatctaccccatatcac gttctcacag ggcagcactc tcatcagaag 120 gaaggagaag aggagtttctccttcacacg tcttgcattt tactgcagga aaataattcc 180 cagaattcgt cactgcagtcgttttcttag atg tca ttg gcc aat ctc tac atc 234 Met Ser Leu Ala Asn LeuTyr Ile 1 5 aat cac tgg caa ggt gaa ctg gaa tct cag cga ttg gct aca gcagtt 282 Asn His Trp Gln Gly Glu Leu Glu Ser Gln Arg Leu Ala Thr Ala Val10 15 20 atg att cat ttg cag cat cag gca cat tgt tac ctg cac aaa agt agg330 Met Ile His Leu Gln His Gln Ala His Cys Tyr Leu His Lys Ser Arg 2530 35 40 att tac tta aac tgg agg aag cag aaa atg cac tcc agt gtt gtt act378 Ile Tyr Leu Asn Trp Arg Lys Gln Lys Met His Ser Ser Val Val Thr 4550 55 caa tgg agt ctg cta caa aag gaa ttc aca ggg gtc cac tct gtt tgt426 Gln Trp Ser Leu Leu Gln Lys Glu Phe Thr Gly Val His Ser Val Cys 6065 70 cag ggt gct ggt ttc ctg tca tta gtt ttc att act ctc ttt cat cct474 Gln Gly Ala Gly Phe Leu Ser Leu Val Phe Ile Thr Leu Phe His Pro 7580 85 aca tgt gtg ggc tca gca act ttg tga attctggttt gtggaatttg 521 ThrCys Val Gly Ser Ala Thr Leu 90 95 acacagatga tttctctgcc aacattgaatctcacaggtc acatcttcca tcctaaccta 581 tatcctaact tttctaccta tcttggcattgtctttgtat tttgccttgt gtaatttgga 641 ctgcactgga tatagtcttt ggtcatatcccctcagcatt aggtgtgggc agcaggcatg 701 gggcatcgtg cattagtaac cagctcagcccacagatgct gtccatgagc ttcctgtccc 761 ctacccttcc ccaaagaagg gcatcagccagttactccca acagtagggt cactcttctc 821 agagaactat tgggattact tacatcgctcggttgttacg cacactacac aacagggaaa 881 cttgttcatt cctccgtccc aaggtttgtcaatctagagt agactgagtg ttcttctgag 941 cttgcaattc acattattgg ctggattcttctggggacat ggtcagagga gctcactatg 1001 gaatctgtgc attaaccact cctgatcctataggctgtat ttaccagctt gttctagcat 1061 cagcacaggt tatggtgata gttgtacagtgaccatatcc aattatcaac atttacctct 1121 ccagcatctg caccaacaga tggacaaataaacaactaag agagaaataa aatgatcctg 1181 ggggcctgtt gtttccctga actatctaccagttgaggga gattgtatgt tcatagggtg 1241 ggagattcca aagcccacct ttctgagcactcataaaata tttgattaca aagggatctt 1301 tgtgcttgag tggtagatgt tggtttgggggctgattaac gagcttagtt agaaaactac 1361 taggtagcca aatgcatgac tgccagattctgttctgtgg attcaaatac tgatctgcaa 1421 tatggcaatg tgactctata gtcagcatactaggaagggt ggttagggct ggtgaagcac 1481 tggtatggac ttaggttcag atcccacttctaaatgctcc ttagtgtctc aaagctttac 1541 tttacctata cgtaaaataa gaatagctttctcataggat tggtgactgg ataaaacaac 1601 ttcgttaaag cac 1614 2 96 PRT Homosapiens 2 Met Ser Leu Ala Asn Leu Tyr Ile Asn His Trp Gln Gly Glu LeuGlu 1 5 10 15 Ser Gln Arg Leu Ala Thr Ala Val Met Ile His Leu Gln HisGln Ala 20 25 30 His Cys Tyr Leu His Lys Ser Arg Ile Tyr Leu Asn Trp ArgLys Gln 35 40 45 Lys Met His Ser Ser Val Val Thr Gln Trp Ser Leu Leu GlnLys Glu 50 55 60 Phe Thr Gly Val His Ser Val Cys Gln Gly Ala Gly Phe LeuSer Leu 65 70 75 80 Val Phe Ile Thr Leu Phe His Pro Thr Cys Val Gly SerAla Thr Leu 85 90 95 3 24 DNA Artificial oligonucleotide primer 3aattattttc ctgcagtggt ttgc 24 4 24 DNA Artificial oligonucleotide primer4 gtgctttaac gaagttgttt tatc 24 5 34 DNA Artificial oligonucleotideprimer 5 ccccatatgg attgatgtag agattggcca atga 34 6 34 DNA Artificialoligonucleotide primer 6 catggatcct cacaaagttg ctgagcccac acat 34 7 15PRT Artificial portion of SEQ ID NO2 7 Met Ser Leu Ala Asn Leu Tyr IleAsn His Trp Gln Gly Glu Leu 1 5 10 15 8 41 DNA Artificialoligonucleotide primer 8 ggcaaggtga actggaatct cagcgattgg ctacagcagt t41 9 41 DNA Artificial oligonucleotide primer 9 ggatcggtga actggaaatcgagcgattgg ctaatcgagt t 41

We claim:
 1. An isolated polypeptide-calcitonin 11-comprising apolypeptide having the amino acid sequence of SEQ ID NO: 2, its activefragments, analogues and derivatives.
 2. The polypeptide of claim 1wherein amino acid sequences of the polypeptide, its analogues orderivatives have at least 95% identity with the amino acid sequence ofSEQ ID NO:
 2. 3. The polypeptide of claim 2 wherein the polypeptide is apolypeptide comprising the amino acid sequence of SEQ ID NO:
 2. 4. Anisolated polynucleotide selected from the group consisting of: (a) thepolynucleotide encoding a polypeptide having an amino acid sequence ofSEQ ID NO: 2 or its fragment, analogue, derivative; (b) thepolynucleotide complementary to polynucleotide (a); and (c) thepolynucleotide sharing at least 70% identity to polynucleotide (a) or(b).
 5. The polynucleotide of claim 4 comprising a polynucleotideencoding an amino acid sequence of SEQ ID NO:2.
 6. The polynucleotide ofclaim 4 wherein the sequence of the polynucleotide comprises position211-501 of SEQ ID NO:1 or position 1-1614 of SEQ ID NO:1.
 7. Arecombinant vector containing an exogenous polynucleotide which isconstructed with the polynucleotide of any of claims 4-6 and plasmid,virus, or expression vector.
 8. A genetically engineered host cellcontaining an exogenous polynucleotide which is selected form the groupconsisting of: (a) the host cell transformed or transfected by therecombinant vector of claim 7; and (b) the host cell transformed ortransfected by the polynucleotide of any of claims 4-6.
 9. A method forproducing a polypeptide having the activity of calcitonin 11, whichcomprises the steps of: (a) culturing the engineered host cell of claim8 under the conditions suitable for expression of calcitonin 11; (b)isolating the polypeptides having the activity of calcitonin 11 proteinfrom the culture.
 10. An antibody specifically which binds boundspecifically with calcitonin
 11. 11. A compound simulating or regulatingthe activity or expression of the polypeptide which is the compoundsimulating, improving, antagonizing, or inhibiting the activity ofcalcitonin
 11. 12. The compound of claim 11 which is an antisensesequence of the polynucleotide sequence of SEQ ID NO: 1 or its fragment.13. The use of the compound of claim 11 for regulating the activity ofcalcitonin 11 in vivo or in vitro.
 14. A method for detecting a diseaserelated to the polypeptide of any of claims 1-3 or susceptibilitythereof which comprises detecting the amount of expression of thepolypeptide, or detecting the activity of the polypeptide, or detectingthe nucleotide variant of the polynucleotide causing the abnormalexpression or activity.
 15. The use of the polypeptide of any of claims1-3 for screening the mimetics, agonists, antagonists or inhibitors ofcalcitonin 11; or for the identification of peptide spectrum.
 16. Theuse of the nucleic acid molecule of any of claims 4-6 wherein it is usedas primer in the nucleic acid amplification, or as probe in thehybridization reaction, or is used for manufacture of gene chip ormicroarray.
 17. The use of the polypeptide, polynucleotide or compoundof any of claims 1-6 and 11 wherein a safe and effective amount of thepolypeptide, polynucleotide or its mimetics, agonists, antagonists orinhibitors are mixed with the pharmaceutically acceptable carrier toform the pharmaceutical composition for the diagnosis or treatment ofdiseases associated with the abnormality of calcitonin
 11. 18. The useof the polypeptide, polynucleotide or compound of any of claims 1-6 and11 wherein the polypeptide, polynucleotide or compound are used for themanufacture of medicine for the treatment of developmental disorders,diseases caused by abnormal metabolism of immune system, and cancers.21. (New) The polypeptide of claim 20 wherein said polypeptide comprisesthe amino acid sequence of SEQ ID NO:
 2. 22. (New) An isolatedpolynucleotide selected from the group consisting of: (a) apolynucleotide encoding a polypeptide that has a calcitonin 11 activityand is at least 95% identical to SEQ ID NO: 2; or (b) a polynucleotidecomplementary to polynucleotide (a).
 23. (New) A polynucleotide of claim22 wherein the polynucleotide encodes an amino acid sequence of SEQ IDNO:2.
 24. (New) A polynucleotide of claim 22 wherein the sequence ofsaid polynucleotide comprises position 211-501 of SEQ ID NO:1.
 25. (New)A polynucleotide of claim 24 wherein the sequence of said polynucleotidecomprises position 1-1614 of SEQ ID NO:1.
 26. (New) A recombinant vectorcomprising a polynucleotide of claim 22, and a suitable regulatoryelement.
 27. (New) A genetically engineered host cell comprising apolynucleotide of claim
 22. 28. (New) A method for producing apolypeptide having an activity of a calcitonin 11 which comprises thesteps of: (a) culturing an engineered host cell of claim 27 underconditions suitable for the expression of a calcitonin 11; and (b)isolating a polypeptides having the activity of a calcitonin 11 from theculture.
 29. (New) An antibody which binds specifically for apolypeptide of claim
 20. 30. (New) A compound simulating, enhancing,antagonizing, or inhibiting a calcitonin 11 activity of a polypeptide ofclaim
 20. 31. (New) A compound of claim 30 which is an antisensesequence of the polynucleotide sequence of SEQ ID NO: 1 or its fragmentwhich inhibits expression of SEQ ID NO:1 in a cell.
 32. (New) A methodfor regulating the activity of a calcitonin 11 in vivo or in vitro,comprising applying a compound of claim 30 to a calcitonin 11 or apolynucleotide encoding a calcitonin
 11. 33. (New) A method fordetecting a disease related to the polypeptide of claim 20, or fordetermining a susceptibility of a mammal thereto, said method comprisingdetecting the amount of expression of said polypeptide, or detecting theactivity of said polypeptide, or detecting the nucleotide variant of apolynucleotide encoding the polypeptide which variant causes saidabnormal expression or activity.
 34. (New) A method for screeningmimetics, agonists, antagonists or inhibitors of a calcitonin 11; or forpeptide profiling, the method comprising labeling a polypeptideaccording to claim 20 and applying the labeled polypeptide in thescreening or profiling.
 35. (New) A method for nucleic acidamplification, or for nucleic acid hybridization reaction, or formanufacture of gene chip or microarray, the method comprisingsynthesizing a polynucleotide of claim
 22. 36. (New) A pharmaceuticalcomposition comprising a polypeptide according to claim 20, and apharmaceutically acceptable carrier.
 37. (New) A method for thetreatment of developmental disorders, diseases caused by abnormalmetabolism of immune system, and cancers wherein the method comprisingadministering an effective amount of the pharmaceutical composition ofclaim 36 to a patient in need thereof.
 38. (New) A pharmaceuticalcomposition comprising a polynucleotide according to claim 22, and apharmaceutically acceptable carrier.